Project Summary/Abstract The long-term goal of this proposal is to better understand how T cells defend against pathogens and eradicate cancerous cells within our bodies. To achieve this goal, T cells continuously crawl seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, then it initiates activation mechanisms that unleash a potent immune response. Malfunctions in T cell activation are linked with autoimmune disease, while drugs that enhance T cell activation are used to treat cancer. Therefore, there is much interest in understanding the molecular mechanisms of T cell activation. The very first step in T cell activation involves recognition between the T cell receptor (TCR) and the short peptides (8-11 amino acids) presented by the major histocompatibility complex (pMHC) protein. Because T cells are highly migratory and antigen recognition occurs when the T cell physically contacts a target cell, there are long standing questions of whether T cells transmit defined forces to their TCR complex and if chemo-mechanical coupling influences immune function. These questions cannot be answered using conventional imaging approaches. The central hypothesis of the proposed work is that advanced mechano- imaging and mechano-analytical approaches will reveal the TCR forces involved in regulation of T cell signaling. Building on our recent breakthroughs in developing high-resolution imaging approaches to map the forces transmitted by cell surface receptors, we will aim to close this gap in our understanding and unravel the mechanical basis of T cell activation. Our preliminary data clearly shows that we have successfully developed the first molecular probes to image the piconewton forces transmitted by the TCR to its ligand during TCR activation. We will test the central hypothesis by first developing molecular force microscopy for the TCR. These probes will test whether the TCR is an anisotropic mechanosensor as proposed in the literature. Next we will map TCR forces within membrane-membrane junctions where the receptor is free to assemble into signaling microclusters. Fluorescence lifetime imaging microscopy (FLIM) and ratiometric probes will be used to map these forces in space and time. Finally, we will use mechanically-triggered enzymes to quantify TCR forces with ultrahigh sensitivity and to tag proximal molecules that are recruited following transmission of TCR forces. The work requires multidisciplinary approaches combining expertise from three investigators that cover the areas of biophysical chemistry, cell biology, and molecular immunology. Importantly, not only will the imaging and quantification techniques developed for this proposal be critical for better understanding the specificity of the adaptive immune system, we expect important implications for the optimal design and implementation of adoptive T cell transfer and chimeric antigen receptors (CARs) in immunotherapy as well as understanding the causes of autoimmune disease.